Recombinant Rhodopirellula baltica NAD (P)H-hydrate epimerase (nnrE)

What is Rhodopirellula baltica NAD(P)H-hydrate epimerase and what is its primary function?

Rhodopirellula baltica NAD(P)H-hydrate epimerase (nnrE) is an enzyme that catalyzes the epimerization of NAD(P)H hydrates. It belongs to the class of isomerases with EC number 5.1.99.6, also known as NAD(P)HX epimerase. The enzyme is derived from the marine bacterium Rhodopirellula baltica strain SH1. Its primary function involves the repair of damaged forms of NAD(P)H by catalyzing the conversion between the two epimeric forms of NAD(P)H hydrates, which is essential for maintaining proper cellular metabolism .

What is the structural composition of Recombinant Rhodopirellula baltica NAD(P)H-hydrate epimerase?

The Recombinant Rhodopirellula baltica NAD(P)H-hydrate epimerase is a full-length protein comprising 244 amino acids (positions 1-244). The amino acid sequence is documented as: MSPEVLSARP GFVRNLMDVM MKPVLQLPPM TCQRIREIDS LAMEQFQMPG IILMENAGRG AAELIEELAP EGNVLILCGK GNNGGDGFTI ARHLQLAGRE VMILAMAATN ELQGDAAIQA KIAEAAGIKI QVVGEAVEAG RLPATDIVVD GLLGTGAKPP LRGRYAEVVE AANASSAIRI ALDIPTGMNG DTGETGETTF RADHTLTFAA PKVGFEKLGA ARFTGEVHVI SIGVPLELLR QFSV . This sequence information is critical for researchers undertaking structural studies or planning mutagenesis experiments.

In which expression systems can Recombinant Rhodopirellula baltica NAD(P)H-hydrate epimerase be produced?

Recombinant Rhodopirellula baltica NAD(P)H-hydrate epimerase can be expressed in multiple heterologous systems, each offering distinct advantages for different experimental applications:

Researchers should select the expression system based on their specific experimental requirements, considering factors such as post-translational modifications, protein solubility, and downstream applications .

What are the optimal conditions for expressing functional Recombinant Rhodopirellula baltica NAD(P)H-hydrate epimerase in E. coli?

Expressing functional Recombinant Rhodopirellula baltica NAD(P)H-hydrate epimerase in E. coli requires careful optimization of several parameters. Based on standard recombinant protein expression protocols, researchers should consider:

• Expression strain selection: BL21(DE3), Rosetta, or Arctic Express strains may improve expression of functional protein.

• Temperature modulation: Lower induction temperatures (16-25°C) often improve solubility and folding.

• Induction parameters: IPTG concentration typically between 0.1-1.0 mM, with induction at mid-log phase (OD600 = 0.6-0.8).

• Media composition: Enriched media such as Terrific Broth or auto-induction media can enhance yields.

• Co-expression with molecular chaperones may increase soluble protein yields.

Post-expression purification typically involves affinity chromatography using the specific tag incorporated during cloning, followed by size-exclusion chromatography to ensure homogeneity. Protein purity should be verified by SDS-PAGE with a target purity of >85% for most applications .

How can researchers effectively use in vivo biotinylation for studying NAD(P)H-hydrate epimerase interactions?

The in vivo biotinylation approach using AviTag-BirA technology provides a powerful tool for researchers studying NAD(P)H-hydrate epimerase interactions. This method involves co-expression of the enzyme tagged with a 15-amino acid AviTag peptide alongside the E. coli biotin ligase (BirA), which catalyzes the site-specific attachment of biotin to the lysine residue within the AviTag sequence .

To implement this approach effectively:

• Design constructs that incorporate the AviTag sequence at either the N- or C-terminus, with appropriate linker sequences to minimize steric hindrance.

• Co-transform expression hosts with both the AviTag-epimerase and BirA expression plasmids.

• Supplement growth media with biotin (10-50 μM) to ensure sufficient substrate availability.

• Verify biotinylation efficiency using Western blot with streptavidin-HRP conjugates or mass spectrometry.

Once biotinylated, the enzyme can be immobilized on streptavidin-coated surfaces for various applications:

• Pull-down assays to identify protein-protein interactions

• Surface plasmon resonance (SPR) studies to determine binding kinetics

• Single-molecule studies using streptavidin-coated quantum dots or surfaces

• Enzyme immobilization for continuous flow catalysis experiments

This approach is particularly valuable for studying the interactions of NAD(P)H-hydrate epimerase with potential protein partners or substrates in its metabolic pathway.

How can site-directed mutagenesis be used to enhance the thermostability of Rhodopirellula baltica NAD(P)H-hydrate epimerase?

Enhancing thermostability of Rhodopirellula baltica NAD(P)H-hydrate epimerase through site-directed mutagenesis requires a systematic approach based on structural analysis. For NAD(P)H-hydrate epimerase and similar enzymes, researchers can employ the following methodological workflow:

• Structural analysis and B-factor identification: High B-factors in protein structures indicate regions of flexibility that often correlate with thermal instability. Using crystallographic data or homology models, identify residues in high-flexibility regions .

• Comparative sequence analysis: Align the sequence with thermostable homologs from thermophilic organisms to identify potentially stabilizing substitutions.

• Rational design strategies:

  • Introduce proline residues in loop regions to reduce flexibility

  • Increase surface salt bridges through charge-charge interactions

  • Optimize hydrophobic core packing

  • Introduce disulfide bonds at strategic positions

• Implementation and validation workflow:

• Iterative optimization: Combine beneficial mutations and test for additive or synergistic effects.

This approach has been successfully applied to similar enzymes, such as D-allulose 3-epimerase from Rhodopirellula baltica, where strategic mutations in flexible regions significantly improved thermostability without compromising catalytic activity .

What analytical techniques are most effective for characterizing the enzymatic activity of Recombinant Rhodopirellula baltica NAD(P)H-hydrate epimerase?

Characterizing the enzymatic activity of Recombinant Rhodopirellula baltica NAD(P)H-hydrate epimerase requires a combination of specialized analytical techniques that can detect the subtle structural changes during the epimerization reaction. The most effective approaches include:

• Spectrophotometric assays: Monitoring changes in absorption spectra at 290-340 nm to detect the conversion between different NAD(P)H hydrate forms. This approach typically employs coupled enzyme assays that link the epimerization reaction to a detectable spectrophotometric change.

• High-Performance Liquid Chromatography (HPLC): Utilizing reverse-phase or ion-exchange chromatography to separate and quantify substrate and product forms. This method allows for direct measurement of reaction progress and can be coupled with mass spectrometry for improved sensitivity.

• Nuclear Magnetic Resonance (NMR) spectroscopy: 1H and 31P NMR can provide detailed structural information about substrate-product interconversion in real-time, allowing for direct observation of the epimerization process.

• Isothermal Titration Calorimetry (ITC): For determining binding constants and thermodynamic parameters of substrate binding.

• Enzyme kinetics analysis: Systematic determination of key kinetic parameters:

These approaches together provide a comprehensive characterization of enzyme activity, substrate specificity, and reaction mechanisms, which are essential for understanding the biological function and potential applications of NAD(P)H-hydrate epimerase.

How can researchers address potential discrepancies in kinetic data when studying NAD(P)H-hydrate epimerase from different expression systems?

When researchers encounter discrepancies in kinetic data for NAD(P)H-hydrate epimerase expressed in different systems (yeast, E. coli, baculovirus, or mammalian cells), a systematic analytical approach is required. The methodology should include:

• Normalization protocols: Establish standard protein quantification methods (e.g., BCA or Bradford assays) and ensure activity data is normalized to protein concentration.

• Expression system comparison matrix:

• Statistical validation: Apply rigorous statistical analysis (ANOVA, t-tests) to determine if observed differences are statistically significant.

• Reconciliation strategies:

  • Develop a standardized assay protocol optimized for all expression systems

  • Use internal controls and reference standards across experiments

  • When possible, express and purify proteins in parallel under identical conditions

  • Document and report all relevant experimental parameters

By implementing this comprehensive approach, researchers can identify whether discrepancies represent genuine biological differences or are artifacts of experimental conditions. This distinction is crucial for accurate interpretation of structure-function relationships in NAD(P)H-hydrate epimerase.

What bioinformatic approaches can help identify potential physiological partners and metabolic pathways involving NAD(P)H-hydrate epimerase?

Identifying the physiological partners and metabolic context of NAD(P)H-hydrate epimerase requires sophisticated bioinformatic approaches. Researchers should implement the following methodological workflow:

• Genomic context analysis:

  • Examine gene clustering in the Rhodopirellula baltica genome to identify co-regulated genes

  • Analyze conserved operons across related species for functional associations

  • Study synteny maps to identify conserved genomic neighborhoods

• Protein-protein interaction prediction:

  • Apply computational methods such as co-evolution analysis to identify potential binding partners

  • Use structural docking simulations to evaluate physical compatibility with candidate partners

  • Explore interactome databases for experimentally verified interactions of homologous proteins

• Metabolic pathway reconstruction:

• Experimental validation design:

  • Plan co-immunoprecipitation or proximity labeling experiments based on bioinformatic predictions

  • Design targeted metabolomic experiments to trace substrate-product relationships

  • Develop genetic knockout or knockdown studies to observe system-wide effects

By integrating these complementary approaches, researchers can develop testable hypotheses about the biological role of NAD(P)H-hydrate epimerase within cellular metabolism and identify key interaction partners for further experimental investigation.

How can researchers leverage various expression tags to optimize purification and functional studies of NAD(P)H-hydrate epimerase?

The selection and implementation of expression tags significantly impact purification efficiency and downstream applications for NAD(P)H-hydrate epimerase. Researchers should consider the following methodological framework:

• Tag selection strategy based on experimental goals:

• Optimization protocol for tag-based purification:

  • Conduct small-scale expression trials with different tag configurations (N-terminal vs. C-terminal)

  • Evaluate tag influence on enzyme activity before committing to large-scale purification

  • Optimize linker length between tag and protein to minimize steric interference

  • For multi-domain proteins, consider domain boundaries when positioning tags

• Application-specific considerations:

  • For structural studies: Use cleavable tags to obtain native protein

  • For immobilization: Consider biotinylation or site-specific chemistry

  • For interaction studies: Select tags unlikely to interfere with binding surfaces

  • For high-throughput screening: Prioritize tags with consistent, reproducible behavior

This systematic approach to tag selection and optimization ensures that researchers can efficiently purify NAD(P)H-hydrate epimerase while maintaining its native functional properties for downstream applications .

What are the most promising approaches for investigating the physiological role of NAD(P)H-hydrate epimerase in cellular metabolism?

Understanding the physiological role of NAD(P)H-hydrate epimerase in cellular metabolism requires an integrated experimental approach that combines genetic, biochemical, and systems biology methods:

• Genetic manipulation strategies:

  • CRISPR-Cas9 gene knockout or knockdown in model organisms

  • Complementation studies with wild-type and mutant variants

  • Construction of conditional expression systems to study time-dependent effects

• Metabolomics approach:

  • Targeted metabolite analysis focusing on NAD(P)H and its derivatives

  • Flux analysis using isotope-labeled precursors

  • Comparative metabolomics between wild-type and epimerase-deficient cells

• Stress response analysis:

• Integrative systems approach:

  • Transcriptomic analysis to identify co-regulated genes

  • Proteomic studies to identify interaction partners and post-translational modifications

  • Mathematical modeling of NAD(P)H metabolism incorporating epimerase activity

By combining these complementary approaches, researchers can develop a comprehensive understanding of how NAD(P)H-hydrate epimerase contributes to cellular redox homeostasis, energy metabolism, and stress response pathways. This multi-faceted investigation provides context for the enzyme's role within the broader metabolic network and identifies potential applications in biotechnology and medicine.

What are the current limitations in NAD(P)H-hydrate epimerase research and promising future directions?

Current research on Rhodopirellula baltica NAD(P)H-hydrate epimerase faces several methodological and knowledge limitations. A comprehensive understanding of these challenges, alongside emerging opportunities, provides valuable direction for researchers in this field:

• Current research limitations:

  • Limited structural information specific to the Rhodopirellula baltica enzyme

  • Incomplete characterization of substrate specificity and physiological relevance

  • Poor thermostability hampering industrial and biotechnological applications

  • Insufficient understanding of the enzyme's role in cellular metabolism and stress response

• Emerging methodological approaches:

• Future research priorities:

  • Development of high-throughput screening methods for evolved variants

  • Investigation of the enzyme's role in redox homeostasis under stress conditions

  • Comparative analysis across different species to understand evolutionary conservation

  • Exploration of potential applications in NAD(P)H regeneration systems

As research progresses, collaborative approaches combining structural biology, enzyme engineering, and systems-level analysis will likely yield significant advances in our understanding of NAD(P)H-hydrate epimerase and its applications. Particular attention should be given to thermostability enhancement through rational design or directed evolution approaches, as this remains a significant limitation for practical applications .